Method for preparing porous composite material

ABSTRACT

This invention relates to a process for producing porous and composite materials comprising steps of: freezing a complex containing at least one calcium salt selected from calcium carbonate, calcium phosphate, and hydroxyapatite and collagen, at least a part of which is gelatinized; and then lyophilizing the resultant. The porous and composite materials obtained by the method of the present invention have large pore diameters, high porosities, and adequate mechanical strengths and biodegradability. Thus, they are suitable for implants such as bone fillers, drug carriers for sustained-release, and the like.

TECHNICAL FIELD

The present invention relates to a process for producing porous andcomposite materials. More particularly, the present invention relates toa process for producing porous and composite materials having large porediameters, high porosities, and mechanical strengths that are suitablefor implants such as bone fillers.

BACKGROUND ART

In the past, bone defects used to be restored by a technique oftransplanting a part of a subject's own bones or a technique ofcomplementing or replacing bones with artificial implants. Suchalternative bone implants were required to have properties such asbioadaptability or bone conductivity and bone inductivity (a property ofincorporating bone tissue and accelerating bone formation) in additionto mechanical properties similar to those of biological bones.Accordingly, porous materials such as porous ceramics used to bepreferably employed as alternative bone implants because they wereeasily penetrated by bone tissues and they had good bone conductivityand bone inductivity.

Currently commercialized porous hydroxyapatite, however, isnonabsorbable. Even though this hydroxyapatite was porous, cells couldnot penetrate pores that did not communicate with the exterior becauseof this nonabsorbability. Accordingly, the final strength was not veryhigh. Also, it was likely to collapse upon moisture absorption duringsurgical operations. Thus, the use thereof in surgical operation, whichrequired strength, was difficult.

Bone fillers of porous β-TCP, which is bioabsorbable and has porosity of75%, are commercialized (Osferion®, Olympus Optical Co., Ltd.; averagepore diameters: 100 μm to 400 μm). This β-TCP is highly likely tocollapse when it is handled. Thus, it was difficult to prepare thismaterial in a form that is adequate for the transplant site, and wasdifficult to handle since implants easily become detached from thetransplant site.

Under the above circumstances, the present inventors have conductedvarious studies to develop a composite material of hydroxyapatite andcollagen that has a structure similar to that of biological bones and toimprove its properties. For example, JP Patent Publication (Kokai) No.7-101708 A (1995) discloses a process for producing a complex of apatiteand an organic substance having the Young's modulus similar to that ofbiological bones with the gradual addition of a mixed solution ofcollagen and phosphoric acid in a suspension of calcium hydroxide. JPPatent Publication (Kokai) No. 11-199209 A (1999) discloses that astructure similar to that of biological bones can be realized byregulating pH and temperature at the time of reaction. Further, JPPatent Publication (Kokai) No. 2000-5298 A discloses a technique forenhancing the formation of apatite on a collagen surface with the use oforganic acid.

In spite of these efforts, however, many complexes of hydroxyapatite andcollagen that were obtained by the conventional technique had small porediameters (approximately 0 μm to 100 μm) and had low porosities (50% orlower).

An object of the present invention is to provide novel porous andcomposite materials having large pore diameters, high porosities, andmechanical strengths that are suitable for implants such as bonefillers.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they have found that compositematerials having large pore diameters and high porosities could beobtained by gelatinizing a part of the collagen constituting a complexof hydroxyapatite and collagen, followed by freezing and lyophilizationthereof. They have also found that application of surface crosslinkingto the composite material realized mechanical strengths that weresuitable for implants such as bone fillers. This has led to thecompletion of the present invention.

More specifically, the present invention provides the following (1) to(12).

(1) A process for producing porous and composite materials comprisingsteps of: cooling a complex containing at least one calcium saltselected from calcium carbonate, calcium phosphate, and hydroxyapatiteand collagen, at least a part of which is gelatinized, to gelate thegelatin; freezing it; and then lyophilizing the resultant.

(2) A process for producing porous and composite materials comprising astep of introducing surface crosslinking between collagens to the porousand composite materials obtained by the process according to (1) above.

(3) The process according to (2) above, wherein the step of introducingsurface crosslinking is carried out by immersing the porous andcomposite materials in a solution containing a crosslinking agent.

(4) The process according to any one of (1) to (3) above comprising astep of adding a crosslinking agent to gelatinized collagen to introduceinternal crosslinking between collagens.

(5) The process according to any one of (1) to (4) above, wherein thecalcium salt is hydroxyapatite.

(6) A process for producing porous and composite materials comprisingthe following steps of:

-   -   1) gelatinizing at least a part of the collagen constituting a        complex containing hydroxyapatite and collagen;    -   2) introducing internal crosslinking between collagens with the        addition of a crosslinking agent to the complex;    -   3) obtaining porous and composite materials by cooling the        complex to gelate the gelatin, freezing it, and then        lyophilizing the resultant; and    -   4) introducing surface crosslinking between collagens by        immersing the porous and composite materials in a solution        containing a crosslinking agent.

(7) The process according to any one of (2) to (6) above, whereininternal and/or surface crosslinking between collagens is introducedusing glutaraldehyde as a crosslinking agent.

(8) The process according to any one of (2) to (7) above, whereinsurface crosslinking between collagens is introduced by immersing theporous and composite materials in a solution containing a crosslinkingagent comprising ethanol as a solvent.

(9) The process according to any one of (1) to (8) above, wherein theresulting porous and composite materials have porosities of 80% orhigher.

(10) The method according to any one of (1) to (9) above using a complexcomprising the c-axis of hydroxyapatite being oriented along collagenfibers.

(11) Porous and composite materials having porosities of 80% or higher,which are produced by the method according to any one of (1) to (10)above.

(12) Implants of the porous and composite materials according to (11)above.

DISCLOSURE OF THE INVENTION

1. A process for producing porous and composite materials

The present invention relates to a process for producing porous andcomposite materials of at least one calcium salt selected from calciumphosphate, calcium carbonate, and hydroxyapatite and collagen.

In the production process according to the present invention, compositematerials having a large number of pores (porous and compositematerials) are produced by cooling a complex containing at least onecalcium salt selected from calcium carbonate, calcium phosphate, andhydroxyapatite and collagen, at least a part of which is gelatinized, togelate the gelatin; freezing it; and then lyophilizing the resultant.Surface crosslinking between collagens is introduced to the resultingporous and composite materials. This allows the porous and compositematerials to have mechanical strengths that are suitable for implants.

The production process according to the present invention is hereafterdescribed in detail.

-   1.1 A complex containing calcium salt and collagen, at least a part    of which is gelatinized

The porous and composite materials according to the present inventionare produced from a complex of at least one calcium salt selected fromcalcium phosphate, calcium carbonate, and hydroxyapatite and collagen,at least a part of which is gelatinized.

1) Calcium salt (hydroxyapatite)

The “calcium salt” that constitutes the complex of the present inventionis at least one selected from calcium phosphate, calcium carbonate, andhydroxyapatite. In particular, hydroxyapatite is the most preferable.

Hydroxyapatite is a compound generally composed of Ca₅(PO₄)₃OH, and itincludes a group of compounds referred to as calcium phosphate such asCaHPO₄, Ca₃(PO₄)₂, Ca₄(PO₄)₂, Ca₁₀(PO₄)₆(OH)₂, CaP₄O₁₁, Ca(PO₃)₂,Ca₂P₂O₇, and Ca(H₂PO₄)₂-H₂O. Also, hydroxyapatite is basically composedof a compound represented by formula Ca₅(PO₄)₃OH or Ca₁₀(PO₄)₆(OH)₂, anda part of the Ca component may be substituted with at least one memberselected from Sr, Ba, MG, Fe, Al, Y, La, Na, K, H, and the like. A partof the (PO₄) component may be substituted with at least one memberselected from VO₄, BO₃, SO₄, CO₃, SiO₄, and the like. A part of the (OH)component may be substituted with at least one member selected from F,Cl, 0, CO₃, and the like. Some of these components may be deficient. Ingeneral, a part of apatite PO₄ and OH components in biological bones aresubstituted with CO₃. Accordingly, the aforementioned components may bepartially substituted by inclusion of CO₃ in the air (about 0% to 10% bymass) during the production of the porous and composite materials of thepresent invention.

Hydroxyapatite is generally microcrystalline, noncrystalline, orcrystalline. Alternatively, it may exist in the form of an isomorphicsolid solution, substitutional solid solution, or interstitial solidsolution. The atomic ratio of calcium/phosphorus (Ca/P) in this“hydroxyapatite” is preferably in the range between 1.3 and 1.8. Inparticular, the range between 1.5 and 1.7 is more preferable. When theatomic ratio is in the range between 1.3 and 1.8, the composition andthe crystal structure of apatite in the product (a calcium phosphatecompound) can be similar to those in bones of vertebrates. This improvesbiocompatibility and bioabsorbability.

2) Collagen

The “collagen” that constitutes the complex of the present inventionincludes a general collagen molecule that has the triple helix structureand gelatinized “denatured collagen,” the triple helix structure ofwhich is destroyed by thermal or other treatment.

At present, about 20 different molecular species of collagens are knownto be present in a wide variety of animal tissues ranging frommammalians to fish. These are generically denoted as “collagens.” Thespecies, the location of tissue, the age, and other factors regardingthe animal that is a starting material for the collagen used in thepresent invention are not particularly limited, and any type of collagencan be used. In general, collagens obtained from skin, bones, cartilage,tendons, organs, or the like of mammalians (such as cow, pig, horse,rabbit, or mouse) and birds (such as chicken) are used. Also,collagen-like proteins obtained from skin, bones, cartilage, fins,scales, organs, or the like of fish (such as cod, left-eyed flounder,right-eyed flounder, salmon, trout, tuna, mackerel, sea bream, sardine,or shark) may be used as starting materials. Alternatively, collagen maybe obtained by gene recombination techniques instead of by extractionfrom animal tissues.

Among the molecular species of collagens, the quantity of type Icollagens is the largest, and they have been well studied. In general,when simple reference is made to a “collagen,” it often indicates type Icollagen. The molecular species of the collagen used in the presentinvention is not particularly limited, and type I collagen is preferablya main component. Further, collagen may be prepared by adequatelysubjecting an amino acid residue of the collagen protein to chemicalmodification such as acetylation, succination, maleylation, phthalation,benzoylation, esterification, amidation, or guanidination.

An example of a process for preparing collagen is extraction from theaforementioned starting material (excluding the gene recombinationtechnique) with the aid of a neutral buffer or dilute acid such ashydrochloric acid, acetic acid, or citric acid. Collagen obtained withthe aid of the former is referred to as neutral salt-soluble collagen,and that obtained with the aid of the latter is referred to asacid-soluble collagen. However, the amount of collagen extracted issmall in either case, and a majority thereof remains as insolublecollagen. Enzyme solubilization and alkali solubilization are known asmethods for solubilizing this insoluble collagen. Collagen obtained bythe former method is referred to as enzyme-solubilized collagen, andthat obtained by the latter method is referred to as alkali-solubilizedcollagen. Both can be solubilized as molecular collagens with yields ofsubstantially 100%.

The method for preparing collagen used in the present invention(extraction type) is not particularly limited. If the molecular weightof solubilized collagen is large, however, the strength of a complexbecomes insufficient because of steric hindrance. Accordingly, the useof monomeric (monomolecular) collagen is preferable. Inenzyme-solubilized collagen and alkali-solubilized collagen, themonomeric collagen content is high, and non-helical regions(telopeptides) having a majority of collagen antigenicity areselectively degraded and removed during the step of production. Thus,they are particularly adequate for the organic or inorganic porous andcomposite materials of the present invention. If these non-helicalregions are degraded and removed from collagen, this collagen isreferred to as “atelocollagen.”

The isoionic point of enzyme-solubilized collagen is different from thatof alkali-solubilized collagen. The isoionic point is the pH level whereboth positive and negative charges, which are derived from a dissociablegroup inherent to a protein molecule, repel each other. In the case ofcollagen, when the pH level approaches the region of the isoionic point,solubilized collagen is known to become fibrous. In general, theisoionic point of the enzyme-solubilized collagen is between pH 8 and 9,and that of the alkali-solubilized collagen is between pH 4 and 5. Inthe present invention, it is preferable to use the enzyme-solubilizedcollagen in a reaction vessel maintained between pH 7 and 11 wherein thefiberization and self-organization is likely to occur. Examples ofenzymes for solubilization include pepsin, trypsin, chymotrypsin,papain, and pronase. Pepsin and pronase are preferably used from theviewpoint of easy handleability after the enzyme reaction.

3) A complex containing calcium salt and collagen

At least a part of the collagen in the complex used in the presentinvention must be gelatinized. The term “gelatin” refers to denaturedcollagen that is obtained by cleavage of salt bonds or hydrogen bondsbetween peptide chains of collagen by treatment with boiling water orthe like, followed by irreversible denaturation into a water-solubleprotein. As mentioned above, the term “collagen” used herein includethis gelatinized collagen. Gelatinization of collagen may be realized bygelatinizing a part of the collagen constituting a complex containingcalcium salt and collagen. Alternatively, gelatinized collagen such as acommercialized solution of gelatin powders may be externally added tothe complex.

In the production process according to the present invention, a “complexcontaining calcium salt and collagen” is employed as a starting materialfor the reaction. Such a complex may be a commercialized one or may beprepared in accordance with a known technique. A preferable example ofthe complex is a “complex of hydroxyapatite and collagen.” Inparticular, a “self-organized complex containing hydroxyapatite andcollagen” is the most preferable. These complexes are described indetail in 2.3 below.

-   1.2 Gelatinization of collagen

Examples of processes for gelatinizing a part of the collagenconstituting the complex containing calcium salt and collagen include amethod wherein a physiological buffer such as PBS or Tris or water isadded to the complex, and the resultant is then heated, and a methodwherein a physiological buffer containing a minor amount of acid orwater is added to the complex, and the resultant is then heated.

The amount of a buffer or water to be added is preferably about 24 to 60ml per g of collagen (in the case of a complex, 25% of which isconstituted by collagen, about 6 to 15 ml of buffer or water per g ofthe complex). By changing the amount of the buffer added, porosities,pore diameters, or strengths of the resulting porous and compositematerials can be adequately controlled. When acid is added, a weak acidsuch as gluconic acid is preferably used together with a weak alkalisuch as calcium carbonate in order to control the pH level. The amountof gluconic acid used is preferably about 0.8 g to 4 g per g of collagen(in the case of a complex, 25% of which is constituted by collagen,about 200 mg to 1,000 mg of gluconic acid per g of the complex). Theamount of alkali that is simultaneously added may be adequatelydetermined in accordance with the amount of gluconic acid in order tobring the pH level to around 5.0.

When acid is not added, the reaction temperature is preferably between40° C. and 50° C., and particularly preferably around 45° C. Thereaction time is preferably about 60 to 120 minutes. When acid is added,the reaction temperature is preferably between 35° C. and 45° C., andparticularly preferably around 40° C. The reaction time is preferablyabout 60 to 120 minutes. That is, collagen can be gelatinized atrelatively low temperature by the method, which is carried out with theaddition of acid.

By changing the reaction temperature or reaction time, the level ofgelatinization can be varied, and the pore diameters or mechanicalstrengths of the resulting porous and composite materials can beadequately controlled. More specifically, if the reaction temperature istoo low or the reaction time is too short, gelatinization isinsufficient. This results in small pore diameters and lowered strengthsof the porous and composite materials. In contrast, if the reactiontemperature is too high or the reaction time is too long, excessivegelatinization occurs. This results in lowered strengths of theresulting porous and composite materials.

-   1.3 Freezing of a complex

Subsequently, a complex containing collagen, at least a part of which isgelatinized, is placed in an adequate mold and then allowed to freeze.

1) Cooling

The aforementioned complex after gelatinization has low viscosity in aheated state. If it is placed in a mold in that state, the complex isprecipitated, and a porous complex having a homogenous structure cannotbe obtained. Thus, the complex is preferably cooled for gelatinizationand then placed in a mold. A method for cooling is not particularlylimited. For example, the complex can be subjected to ice cooling, andcooling is continued until it becomes a jell or mousse.

During this step of cooling, gelatinized collagen is homogenouslyincorporated in a complex and solidified as a jell. Freezing thereofresults in homogenous growth of ice crystals in the complex. These icecrystals become pores in the subsequent step of lyophilization. Thus,porous and composite materials having large pore diameters, highporosities, and specific mechanical strengths (elasticity) can beprovided.

2) Internal crosslinking

Before the complex is placed in a mold, an adequate crosslinking agentis added to the complex in order to introduce internal crosslinkingbetween collagens constituting the complex. This is preferable since theresulting porous body has larger pore diameters and a larger number ofopen pores.

The term “internal crosslinking” used herein refers to crosslinking thatis introduced between collagens that are present in the complex.Crosslinking occurs either between ungelatinized collagens, betweengelatinized collagens, or between ungelatinized collagen and gelatinizedcollagen. Crosslinking may occur at any portions of collagens.Particularly preferable crosslinking occurs between a carboxyl group anda hydroxyl group, between a carboxyl group and a ε-amino group, orbetween ε-amino groups.

Any method, such as chemical crosslinking using a crosslinking agent orcondensing agent or physical crosslinking using γ rays, ultravioletrays, thermal dehydration, an electron beam, or the like, may beemployed. Chemical crosslinking using a crosslinking agent ispreferable.

Examples of crosslinking agents that can be used include: aldehydecrosslinking agents such as glutaraldehyde or formaldehyde; isocyanatecrosslinking agents such as hexamethylene diisocyanate; carbodiimidecrosslinking agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; polyepoxycrosslinking agents such as ethylene glycol diethyl ether; andtransglutaminase, with glutaraldehyde being preferable. The amount ofthe crosslinking agent to be added is adequately determined inaccordance with its type. In the case of glutaraldehyde, it ispreferably 0.4 mg to 40 mg, and particularly preferably about 4 mg to 10mg, per g of collagen before gelatinization (initial amount) (in thecase of a complex, 25% of which is constituted by collagen, 0.1 mg to 10mg, and particularly about 1 mg to 2.5 mg, per g of the complex).

3) Freezing

The cooled complex is allowed to freeze in accordance with aconventional technique. The freezing speed in a freezer is slowed inorder to allow ice crystals to grow. These ice crystals become pores inthe subsequent step of lyophilization. The pore sizes affect thestrengths of the resulting porous and composite materials. Thus, the useof a refrigerator, the temperature of which can be subtly controlled, ismore preferable from the viewpoint of the controllability of thefreezing speed.

-   1.4 Lyophilization

The frozen complex is allowed to lyophilize in accordance with aconventional technique. During this step of lyophilization, numerous icecrystals, which were formed in the complex during the step of freezing,become pores. Thus, porous and composite materials having desirableporosities and pore diameters are obtained.

-   1.5 Surface crosslinking

The mechanical strengths of the resulting porous and composite materialscan be enhanced by introducing surface crosslinking between collagens.The term “surface crosslinking” refers to crosslinking that isintroduced between collagens that are present on the surface of theporous and composite materials.

Any method, such as chemical crosslinking using a crosslinking agent orcondensing agent or physical crosslinking using γ rays, ultravioletrays, thermal dehydration, an electron beam, or the like, may beemployed for surface crosslinking. Examples of crosslinking agentsinclude: aldehyde crosslinking agents such as glutaraldehyde orformaldehyde; isocyanate crosslinking agents such as hexamethylenediisocyanate; carbodiimide crosslinking agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; polyepoxycrosslinking agents such as ethylene glycol diethyl ether; andtransglutaminase. The amount of the crosslinking agent to be used isadequately determined in accordance with its type. It is preferably atleast 1 μmol per g of the complex.

Crosslinking as mentioned above may occur at any portions of collagens.Particularly preferable crosslinking occurs between a carboxyl group anda hydroxyl group, between a carboxyl group and a ε-amino group, orbetween ε-amino groups. Crosslinking is preferably introduced in atleast 1%, and more preferably at least 5%, of reactive functionalgroups. This is because sufficient mechanical strengths cannot beexpected if crosslinking is insufficient. Attention should be given tothe use of a crosslinking agent since the excessive use of acrosslinking agent disadvantageously embrittles a complex.

Among the aforementioned methods for crosslinking, chemical crosslinkingusing a crosslinking agent such as glutaraldehyde is particularlypreferable from the viewpoints of controllability of the level ofcrosslinking and bioadaptability of the resulting complex. Chemicalcrosslinking can be performed by immersing the porous and compositematerials obtained in the section above in a solution containing acrosslinking agent.

A method for crosslinking using glutaraldehyde is hereafter described asa preferable embodiment of the present invention. At the outset, theporous and composite materials obtained in the section above areimmersed in a solution containing 0.001% to 1%, and preferably about0.005% to 0.1%, glutaraldehyde (degassing and aspiration). Use of 50% to100% ethanol as a solvent is preferable since crosslinking density isenhanced. Conditions for immersion are adequately determined inaccordance with the concentration of glutaraldehyde. Preferably, it isgenerally slowly carried out over the course of a period ofapproximately 60 minutes to 5 hours at room temperature or lowertemperature in order to allow the crosslinking to homogenously occur.After the crosslinking, the porous and composite materials are washedwith pure water to remove excess glutaraldehyde. Further, remainingglutaraldehyde may be preferably neutralized with the use of a glycinesolution or the like. The porous and composite materials may be furtherlyophilized after crosslinking.

Thus, the porous and composite materials can be provided with desirablemechanical strengths by introducing surface crosslinking therein. Thedegradation rate in an organism can be controlled based on the level ofcrosslinking to be introduced.

-   1.6 Others

In addition, adequate modifications or additional steps can be added tothe aforementioned steps within the scope of the present invention. Forexample, addition of polysaccharides such as hyaluronic acid prior tothe crosslinking can increase the points of crosslinking and enhance themechanical strengths. Alternatively, polysaccharides such as hyaluronicacid can be added instead of gelatinized collagen, crosslinking can beintroduced thereto, and the resultant can be allowed to gelatinize andfreeze. Thus, a porous body having large pore diameters and highporosities can be obtained.

A wide variety of treatments can be applied in order to enhance thestrengths of the porous and composite materials after surfacecrosslinking. For example, the surfaces of the porous and compositematerials may be coated with hydroxyapatite and collagen by immersingthem in a slurry of a complex of hydroxyapatite and collagen. Thesurfaces of the porous and composite materials may be coated withhydroxyapatite by immersing them in a slurry of hydroxyapatite.Alternatively, hydroxyapatite may be precipitated not only on thesurfaces but also on the insides of the porous and composite materialsby alternate immersion in calcium and phosphorus.

-   2. The porous and composite materials of the present invention-   2.1 Properties of the porous and composite materials (porosities,    pore diameters, and mechanical strengths)

The porous and composite materials obtained by the method according tothe present invention have high porosities, large pore diameters, andadequate mechanical strengths. The porosities of the porous andcomposite materials obtained under preferable conditions are 80% to 95%,and the pore diameters thereof are about 50 μm to 2,000 μm. However, theporosities or pore diameters of the porous and composite materials canbe adequately changed by altering the amount of a buffer, the reactiontemperature, and the reaction time in the step of gelatinization, thefreezing speed in the step of freezing, or other factors. Accordingly,porous and composite materials having desirable porosities and porediameters can be obtained by changing the aforementioned conditions.Further, conditions for internal or surface crosslinking can beadequately determined to obtain porous and composite materials havingdesirable strengths.

-   2.2 Evaluation of properties of porous and composite materials

The porosities and the pore diameters of the porous and compositematerials of the present invention can be measured and evaluated by, forexample, image analysis of electron micrographs. Mechanical strengthscan be evaluated based on, for example, the three-point bending strengthor the Young's modulus, determined based thereon. Alternatively, thestrength can be evaluated based on functional analysis of operabilitywhen it is actually used, such as during transplantation tests.

-   2.3 Self-organized porous and composite materials containing    hydroxyapatite and collagen

A preferable example of the porous and composite material of the presentinvention is a microporous and composite material containinghydroxyapatite and collagen in which the c-axis of hydroxyapatite isoriented along with collagen fiber.

This composite material is produced using a self-organized complexcontaining hydroxyapatite and collagen as a starting material. The term“self-organized” generally refers to the “formation of a specificorganization through the aggregation of homologous or heterologousatoms, molecules, fine particles, or the like by non-covalent bindinginteraction” (Seikagaku Jiten (Dictionary of Biochemistry), Tokyo KagakuDozin Co., Ltd.). In the present invention, this term particularlyrefers to the fact that hydroxyapatite having an apatite structure formsan orientation that is specific to biological bones along collagenfibers. That is, a microporous structure in which the c-axis ofhydroxyapatite is oriented along collagen fibers is formed.Self-organized complexes containing hydroxyapatite and collagen can beproduced in accordance with, for example, the method of Kikuchi et al.(Kikuchi, S. et al., J. Biomater., 22 (13)(2001), 1705-1711, S. Itoh etal., J. Biomed Mater Res. (2001), 445-453).

This microporous structure in which the c-axis of hydroxyapatite isoriented along collagen fibers is partially maintained during theproduction step according to the present invention. Accordingly, theresulting porous and composite materials have microporous structuressimilar to those of biological bones.

-   2.4 Others

The porous and composite materials of the present invention can containother components within the scope of the present invention. Examples ofsuch components include inorganic salts such as St, Mg, and CO₃, organicsubstances such as citric acid and phospholipids, Bone MorphogeneticProteins, and agents such as an anti-cancer agent.

-   3. A method for utilizing porous and composite materials-   3.1 Implants

The porous and composite materials obtained by the method of the presentinvention have high porosities, and each component thereof isbiodegradable. Accordingly, they are easily penetrated by cells and theyare excellent in bone conductivity because they contain hydroxyapatiteand collagen. Further, adequate mechanical strengths and retentivity inorganisms (adequate rate of biodegradation) can be controlled bycrosslinking. Accordingly, the porous and composite materials of thepresent invention are suitable for implants that are used in the fieldof orthopedics such as bone fillers or implants that are used in thefield of dentistry.

The configurations and forms of the aforementioned implants are notparticularly limited. Implants can take any desired configurations andforms in accordance with their applications. For example, they can beblocks, pastes, films, particles, or sponges.

The porous and composite materials of the present invention have lowcompressive strengths. Upon moisture absorption, they become elastic assponges, they are less likely to collapse even with the application offorces strong enough to deform (crush) them, they reabsorb moisture, andthey rapidly regain their original forms. This feature results in anexcellent operability at the time of surgical operations. When theporous and composite materials of the present invention are used asimplants by making use of the aforementioned feature, they may be onceimmersed in an adequate liquid such as physiological saline before use.

-   3.2 Drug carrier for sustained-release (cell carrier, DNA carrier)

The porous and composite materials of the present invention have largesurface areas of hydroxyapatite or the like that are capable ofadsorbing a protein, DNA, or the like and the densities thereof are nottoo high. Thus, penetration and release of an agent or the like caneasily occur. Accordingly, the porous and composite materials of thepresent invention can be used as a drug carrier for sustained-release.Drug carriers for sustained-release using the porous and compositematerials of the present invention have high drug adsorptivity, andtheir forms are well preserved. The aforementioned drug carriers forsustained-release have higher drug-adsorptivity and penetrability bycells compared with a conventional block of hydroxyapatite and collagen.The drug carriers for sustained-release using the porous and compositematerials of the present invention can prevent carcinoma recurrence andinduce the generation of hard tissue of the organism by, for example,impregnating an anti-cancer agent or the like therein to use it forreconstructing bones resected due to osteogenic sarcoma or otherreasons.

The aforementioned carriers can be used as carriers not only for drugsbut also for cells or DNA. For example, when they are used as cellcarriers, cells are introduced into the carrier under negative pressureby suction, and the resultants are applied to a site of interest.

-   3.3 Media for cell culture (scaffold)

The porous and composite materials of the present invention can be usedas media for cell culture (scaffold) through the utilization of theirhigh porosities, large pore diameters, and flexibilities. For example,highly bioactive cytokines are incorporated, the resultant is used asmedia to conduct tissue culture in an environment similar to that oforganisms to which force, electricity, or the like is applied or invivo. Thus, tissues such as bone marrow or liver tissues can bereconstructed.

More specifically, the porous and composite material obtained by thepresent invention can be put to a wide range of applications. Examplesthereof include artificial bones, artificial joints, cements for tendonsand bones, dental implants, percutaneous terminals for catheters, drugcarriers for sustained-release, cell carriers, DNA carriers, media forcell culture, chambers for bone marrow induction, and chambers or basematerials for tissue reconstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs of the HAp/Col porous body prepared in Example1 (A: a complete photograph of the porous body; B: an enlargedphotograph).

FIG. 2 shows photographs of the porous body prepared when the incubationtime in the step of gelatinization is 30 minutes or 90 minutes (A: 30minutes; B: 90 minutes).

FIG. 3 shows photographs of the porous body prepared when the amount ofPBS added in the step of gelatinization is 5 ml or 7 ml (A: 5 ml; B: 7ml).

FIG. 4 shows photographs of the porous body prepared by mixingungelatinized HAp/Col (A: a complete photograph of the porous body; B:an enlarged photograph).

FIG. 5 shows photographs of the porous body prepared when the amount ofGA added in the step of internal crosslinking is 2 μl or 5 μl (A: 2 μlof 25% GA; B: 5 μl of 25% GA).

FIG. 6 shows photographs of the porous body prepared when the freezingtemperature is −20° C. or −80° C. (A:−20° C.; B:−80° C.).

FIG. 7 shows images of HE staining one week after the transplantation of“an HAp/Col porous body that is surface-crosslinked with 0.01% GA andinternally crosslinked” to a rat thighbone in Example 3.

FIG. 8 shows images of HE staining three weeks after the transplantationof “an HAp/Col porous body that is surface-crosslinked with 0.01% GA andinternally crosslinked” to a rat thighbone in Example 3.

FIG. 9 shows images of HE staining two weeks after the transplantationof “an HAp/Col porous body that is surface-crosslinked with 0.01% GA andinternally crosslinked” to a rat thighbone in Example 3.

FIG. 10 shows images of HE staining four weeks after the transplantationof “an HAp/Col porous body that is surface-crosslinked with 0.01% GA andinternally crosslinked” to a rat thighbone in Example 3.

FIG. 11 shows images of HE staining six weeks after the transplantationof “an HAp/Col porous body that is surface-crosslinked with 0.01% GA andinternally crosslinked” to a rat thighbone in Example 3.

FIG. 12 shows images of HE staining six weeks after the transplantationof “an HAp/Col porous body that is surface-crosslinked with 0.05% GA” toa rat thighbone in Example 3.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto.

EXAMPLE 1 Preparation of a Hydroxyapatite/Collagen (HAp/Col) Porous Body

1. Production of HAp/Col Composite Materials

(1) Gelatinization of Collagen

In accordance with the method of Kikuchi et al. (M. Kikuchi et al.,Biomater., 22 (13)(2001), 1705-1711), 500 mg of HAp/Col complex powders(HAp:Col=3:1, lyophilized powders) were prepared. PBS (6 ml) was addedthereto, followed by mixing. The resultant was incubated at 45° C. for90 minutes, and a part of the collagen was allowed to gelatinize.

(2) Internal Crosslinking

The gelatinized HAp/Col complex was allowed to cool. When the complexbecame runny, a mixture of 2 μl of 25% glutaraldehyde with 0.5 ml of PBSwas added thereto, and the resultant was placed in a mold, followed byice cooling.

(3) Freezing

The ice-cooled HAp/Col complex was kept in the mold, placed in a 450 mlplastic case in that state, and then sealed. The resultant was allowedto freeze in a freezer at −20° C.

(4) Lyophilization

The frozen HAp/Col complex was lyophilized in accordance with aconventional technique. The resulting porous body is shown in FIG. 1.

(5) Surface Crosslinking

The lyophilized complex was immersed in 50% ethanol containing 0.01%glutaraldehyde for 2 hours to introduce crosslinking between collagenson the surface of the porous body (surface crosslinking). After thereaction, the complex was washed with pure water to remove remainingglutaraldehyde. A glycine solution was further added thereto toneutralize the remaining glutaraldehyde. Thereafter, the complex waswashed with pure water and allowed to dry to obtain the HAp/Col porousbody of interest.

2. Evaluation of the HAp/Col Porous Body

As is apparent from FIG. 1, the resulting HAp/Col porous body containednumerous bubbles having relatively large sizes (pore sizes:approximately 50 to 2,000 μm). The porosities thereof were deduced to beapproximately 80% to 95%. The surface-crosslinked HAp/Col porous bodybecame an elastic sponge upon immersion in water. Even though it wasdeformed with the application of external force, it could rapidly regainits original form when the force was relieved.

EXAMPLE 2 Examination of Conditions for Producing the HAp/Col PorousBody

Conditions in the production step in Example 1 were varied, and the porediameters and the porosities of the resulting porous bodies wereexamined. These porous bodies were not subjected to surfacecrosslinking.

1. Examinations of conditions for gelatinization

(1) Incubation Time

The HAp/Col porous body was prepared in the same manner as in Example 1except that the incubation time in the step for gelatinization waschanged to 30 minutes. The resultant was compared with the HAP/Colporous body obtained in Example 1 (FIG. 2).

As a result, the porous body that was prepared with an incubation timeof 30 minutes had very small pore diameters compared with that preparedwith an incubation time of 90 minutes.

(2) The Amount of PBS

The HAp/Col porous body was prepared in the same manner as in Example 1except that the amount of PBS added in the step of gelatinization waschanged to 5 ml or 7 ml. The resultant was compared with the HAP/Colporous body obtained in Example 1 (FIG. 3).

As a result, a porous body having sufficiently large pore diameters wasobtained with the use of 5 ml, 6 ml, or 7 ml of PBS. When the amount ofPBS was 7 ml, the pore diameter was slightly larger than that obtainedwith the use of 5 ml or 6 ml of PBS. The porous body was more flexiblewhen it was immersed in water.

(3) Mixing of Ungelatinized HAp/Col

In the step of gelatinization, PBS was added to 30% of HAp/Col (500 mg),and the resultant was incubated at 45° C. for 90 minutes. PBS was addedto the remaining 70% thereof, and the resultant was incubated at 40° C.for 60 minutes (gelatinization hardly occurred at 40° C. for 60minutes). Thereafter, these were mixed with each other to prepare theHAp/Col porous body in the same manner as in Example 1 (FIG. 4).

As a result, a porous body having sufficient pore diameters was obtainedby this method. This porous body had a larger number of continuous poresthan the porous body obtained in Example 1.

(4) Addition of Acid

In the step of gelatinization, 300 mg of gluconic acid and 83 mg ofcalcium carbonate were added to 4 ml of PBS, and the resultant wasincubated at 40° C. for 90 minutes. The porous body was prepared in thesame manner as in Example 1 except for the above-stated condition.

As a result, gelatinization occurred at 40° C. when acid was added, anda porous body having larger pore diameters was obtained.

2. Examination of Conditions for Internal Crosslinking

The HAp/Col porous body was prepared in the same manner as in Example 1except that the amount of 25% glutaraldehyde added in the step ofinternal crosslinking was changed to 5 μl. The resultant was comparedwith the HAP/Col porous body obtained in Example 1 (FIG. 5).

As a result, the porous body prepared with the addition of 5 μl of 25%glutaraldehyde and then immersed in water was harder and had a largernumber of continuous pores compared with the porous body prepared withthe addition of 2 μl of 25% glutaraldehyde and similarly immersed inwater.

4. Examination of Conditions for Freezing

The HAp/Col porous body was prepared in the same manner as in Example 1except that the HAp/Col complex was allowed to freeze in a freezer at−80° C. without being placed in a case in the step of freezing. Theresultant was compared with the HAP/Col porous body obtained in Example1 (FIG. 6).

As a result, the porous body prepared by freezing at −80° C. had verysmall pore diameters compared with that prepared by freezing at −20° C.

Example 3 Experimentation for Transplanting the HAp/Col Porous Body to aRat Thighbone

1. Preparation of Implants for Transplantation

In accordance with the method of Kikuchi et al., 500 mg of HAp/Colcomplex powders (HAp:Col=3:1) were prepared. PBS (4 ml), 300 mg ofgluconic acid, and 83 mg of calcium carbonate were added thereto, andthe mixture was incubated at 40° C. for 90 minutes for gelatinization.The gelatinized complex was allowed to freeze in a freezer at −20° C.,and further lyophilized to obtain the HAp/Col porous body. The resultingporous body was immersed in a solution of 0.01% glutaraldehyde for 2hours to introduce crosslinking between collagens on the surface of thecomplex (surface crosslinking).

Porous bodies were prepared under the above conditions. One type thereofwas provided with internal crosslinking in the same manner as in Example1, and another type was provided with surface crosslinking introducedwith 0.5% glutaraldehyde (GA).

The prepared porous bodies were cut into 2×2×3 mm portions to prepareimplants to be transplanted into rat thighbones.

2. Transplantation into rat thighbones

A hole (diameter: approximately 3 mm) was provided in the distal part ofa Wistar rat thighbone, and the prepared implants were transplantedthereinto. The implants were taken out 1, 2, 3, 4, and 6 weeks after thetransplantation and stained with hematoxylin and eosin for observation(FIGS. 7 to 12).

3. Results

(1) HAp/Col Porous Body Surface-crosslinked with 0.01% GA

This porous body had lower porosity and a smaller number of continuouspores compared with the porous body of (2), which was subjected tointernal crosslinking. Thus, penetrability by cells was less sufficient.With the elapse of time, however, implants were absorbed, and good boneformation was observed.

One week after the transplantation, good cell penetration in thevicinity or formation of neonatal bones was observed in a part thereof(FIGS. 7B, 7C). In other parts, there were pores which cells did notpenetrate (FIG. 7A). Cell penetration was enhanced 2 weeks after thetransplantation, and formation of neonatal bones that were in contactwith the implants was observed. Three weeks after the transplantation,cell penetration and formation of neonatal bones were further enhancedin parenchyma (FIGS. 8A, 8C), and implants were significantly decreasedbecause of absorption as neonatal bones were formed (FIG. 8B).

(2) HAp/Col Porous Body Surface-crosslinked with 0.01% GA and InternallyCrosslinked

The porous body to which internal crosslinking had been applied hadsomewhat larger pore diameters and the penetration by cells was bettercompared with the porous body of (1). While good cell penetration andformation of neonatal bones around the implants were observed 2 weeksafter the transplantation (FIG. 9A), absorption of implants byosteoclasts was observed (FIG. 9B). Four weeks after thetransplantation, absorption of implants and neonatal bones was observed(FIG. 10A to 10C). It was observed that absorption of implants andneonatal bones was further enhanced 6 weeks after the transplantation(FIG. 11A to 11C).

(3) HAp/Col Porous Body Surface-crosslinked with 0.05% GA

This porous body had frequent crosslinking. Thus, absorption of implantswas poor, and implants still remained even 6 weeks after thetransplantation (FIG. 12D).

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention enables the production of biodegradable porous andcomposite materials which have large pore diameters, high porosities,and mechanical strengths that are suitable for alternative boneimplants. The porous and composite materials produced by the presentinvention are very useful as implants, various carriers such as drugcarriers for sustained-release, and supports for cell culture.

1. A process for producing porous and composite materials comprisingsteps of: cooling a complex containing at least one calcium saltselected from calcium carbonate, calcium phosphate, and hydroxyapatiteand collagen, at least a part of which is gelatinized, to gelate thegelatin; freezing it; and then lyophilizing the resultant.
 2. A processfor producing porous and composite materials comprising a step ofintroducing surface crosslinking between collagens to the porous andcomposite materials obtained by the process according to claim
 1. 3. Theprocess according to claim 2, wherein the step of introducing surfacecrosslinking is carried out by immersing the porous and compositematerials in a solution containing a crosslinking agent.
 4. The processaccording to any one of claims 1 to 3 comprising a step of adding acrosslinking agent to gelatinized collagen to introduce internalcrosslinking between collagens.
 5. The process according to any one ofclaims 1 to 3, wherein the calcium salt is hydroxyapatite.
 6. A processfor producing porous and composite materials comprising the followingsteps of: 1) gelatinizing at least a part of the collagen constituting acomplex containing hydroxyapatite and collagen; 2) introducing internalcrosslinking between collagens with the addition of a crosslinking agentto the complex; 3) obtaining porous and composite materials by coolingthe complex to gelate the gelatin, freezing it, and then lyophilizingthe resultant; and 4) introducing surface crosslinking between collagensby immersing the porous and composite materials in a solution containinga crosslinking agent.
 7. The process according to claim 2 or 3, whereininternal and/or surface crosslinking between collagens is introducedusing glutaraldehyde as a crosslinking agent.
 8. The process accordingto any one of claims 1 to 3, wherein surface crosslinking betweencollagens is introduced by immersing the porous and composite materialsin a solution containing a crosslinking agent comprising ethanol as asolvent.
 9. The process according to any one of claims 1 to 3, whereinthe resulting porous and composite materials have porosities of 80% orhigher.
 10. The method according to any one of claims 1 to 3 using acomplex comprising the c-axis of hydroxyapatite being oriented alongcollagen fibers.
 11. Porous and composite materials having porosities of80% or higher, which are produced by the method according to any one ofclaims 1 to
 3. 12. Implants of the porous and composite materialsaccording to claim 11.